ABSTRACT Borrelia miyamotoi is a tick-transmitted spirochete recently shown to cause infection and disease in the United States, Europe and Asia. A member of the relapsing fever group of Borrelia, this pathogen has been proven to cause most commonly a nonspecific, flu-like illness with fever, fatigue, myalgia, and headache. Disease can be particularly severe and involve the central nervous system in immunocompromised patients. The true extent of disease due to this bacterium is difficult to assess because of the lack of a fully validated diagnostic assay. Conversely, development of a clinically useful test is difficult because of the lack of a large cohort of clinically well-characterized patients infected with B. miyamotoi. We have access to two populations of well-characterized patients from which we can identify cases of active disease. Our goal over Phases I and II of this project is to assemble a collection of up to 100 serum samples from patients known to have been exposed to and/or infected with B. miyamotoi. The first cohort of patients consists of individuals who have been bitten by ticks and who have submitted these ticks for analysis to our collaborator Dr. Rich. If the tick is found to be infected with Borrelia miyamotoi, we will contact the patient and obtain blood and serum samples. The second group of patients will consist of individuals who consulted their healthcare professional with symptoms of febrile illness in the spring and summer and who are suspected of having a tickborne illness. We have developed a prototype multiplexed serological assay for Borrelia miyamotoi infection based on seven published or proprietary antigens. We propose to validate this assay by analyzing serum samples from both cohorts of infected patients. Statistical methods will be used to identify the most useful diagnostic antigens and to develop an algorithm optimizing the sensitivity and specificity of the assay. Patients infected with Borrelia miyamotoi will be identified by a PCR reaction that is positive for this pathogen, or by evidence of seroconversion between acute and convalescent samples. A statistical approach will allow us to identify those antigens that are diagnostically informative and to develop validated criteria to correctly diagnose B. miyamotoi infection using our multiplex assay.